KR102248581B1 - Interference-suppressed immunoassay to detect anti-drug antibodies in serum samples - Google Patents

Abstract

The present invention reports an enzyme-linked immunosorbent assay for detection of an anti-drug antibody against a drug antibody in a sample containing a capture drug antibody and a tracer drug antibody, wherein 0.5 μg of the capture drug antibody and the tracer drug antibody /Ml or more, and the sample is incubated for 1 to 24 hours simultaneously with the capture drug antibody and the tracer drug antibody, the capture drug antibody and the tracer drug antibody are derivatized through a single lysine residue, and the The sample contains 10% serum, and oligomeric human IgG is added to the sample prior to incubation with the capture drug antibody and the tracer drug antibody.

Description

INTERFERENCE-SUPPRESSED IMMUNOASSAY TO DETECT ANTI-DRUG ANTIBODIES IN SERUM SAMPLES for detecting anti-drug antibodies in serum samples

The present invention reports interference-suppressed immunosuppression and its use for detecting anti-drug antibodies in serum samples of patients treated with anti-inflammatory antibodies.

Clinical development of novel therapeutic antibodies requires evaluation of the potential immunogenicity of these antibodies by appropriate assays (Kaliyaperumal, A. and Jing, S., Curr. Pharm. Biotechnol. 10 (2009) 352-358) ]). Anti-drug antibody (ADA) testing usually involves two sets of approaches: (1) assays for ADA detection and (2) assays for ADA characterization. ADA detection assays include screening and specificity confirmation (confirmation) assays. Microtiter plate-based enzyme-linked immunosorbent assay (ELISA) is still the most widely used format for screening ADA due to its high processing efficiency, relative simplicity and high sensitivity (Geng, D., et al. ., J. Pharm. Biomed. Anal. 39 (2005) 364-375]). ADA ELISA is most commonly designed as a bridging format that provides high selectivity, detection of all isotypes and ability to detect pan-species ADA (Mire-Sluis, AR, et al., J. Immunol. Methods 289 (2004). 1-16]).

A crosslinked ELISA was developed and used as a screening and confirmation ADA assay for the anti-IL6R antibody tocilizumab (Stubenrauch, K., et al., Clin. Ther. 32 (2010) 1597-1609).

Stubenrauch, K. et al. report a general anti-drug antibody assay for drug resistance in serum samples from mice exposed to approved antibodies (Anal. Biochem. 430 (2012) 193-199). ]). Bourdage (JS) et al. report the effect of a dual antigen bridging immunoassay format on antigen coating concentration dependence and relevance to design immunogenicity assays for monoclonal antibodies (J. Pharm. Biochem. Anal. 39 (2005) 685-690]). Mikulskis, A. et al. report solution ELISA as a selection criterion for the development of robust, drug-resistant immunogenicity assays in support of drug development (J. Immunol. Meth. 365 (2010) 38- 49]).Pan, J. et al. report a comparison between the ELISA method and the NIDSA® high-speed assay in immunogenicity tests of two biotherapy regimens (J. Pharm. Tox. Meth. 63). (2010) 150-159]).

Identification analysis is reported in WO 2009/077127.

Qiu, ZJ et al. report a novel allogeneic biotin-digoxigenin based assay for detection of human anti-therapeutic antibodies in autoimmune serum (J. Immunol. Meth. 362 (2010) 101-111) ]).

The present invention reports a cross-linked enzyme-linked immunosorbent assay (cross-linked ELISA) that can be used as a screening, identification and follow-up assay for the detection of anti-drug antibodies (ADA) in serum-containing samples of patients treated with therapeutic antibodies. The assay as reported herein is particularly useful if the serum containing sample is from a patient with an autoimmune disease such as rheumatoid arthritis (RA).

As reported herein, the assay shows improved resistance to the amount of therapeutic antibody in the sample to be analyzed (increased drug resistance of ADA ELISA) and at the same time reduces the number of false positive assay results.

It has been found that interference by free drugs and rheumatoid factors (RF) can be minimized by assays as reported herein.

This assay is particularly useful if the sample contains antibodies other than the anti-drug antibody in question that can interfere with immunoassays for detection of anti-drug antibodies and thus may cause false positive immunoassay results.

In one embodiment, the method as reported herein is used for the determination of anti-drug antibodies of drug antibodies used in anti-inflammatory therapy.

The increased drug resistance results from 1) increasing the concentration of biotinylated and digoxigenylated capture and tracking reagents; 2) simultaneous cultivation of the capture and tracking reagent and the serum sample instead of continuous cultivation; 3) extended incubation time; 4) the use of uniformly single-coupled capture and tracking reagents; And 5) the use of increased serum substrate content.

Interference from rheumatoid factors can be suppressed by the addition of oligomeric human immunoglobulin G (IgG) as an additive.

The drug resistance of the interference-suppressed ADA assay as reported herein is at least 10 times higher than that of assays known in the art.

One aspect as reported herein is an enzyme-linked immunoabsorption assay for detection of anti-drug antibodies against drug antibodies in a sample comprising a capture drug antibody and a tracer drug antibody, wherein

a) using the capture drug antibody and the tracer drug antibody at a concentration greater than 0.5 μg/ml,

b) incubating the sample simultaneously with the capture drug antibody and the tracer drug antibody for 4 to 24 hours,

c) derivatizing the capture drug antibody and the tracer drug antibody through a single lysine residue,

d) the sample contains at least 7.5% serum,

e) Oligomeric human IgG is added to the sample prior to incubation with the capture drug antibody and the tracer drug antibody.

One aspect as reported in the present invention is an enzyme-linked immunosorbent assay for detection of an anti-drug antibody against a drug antibody in a sample of a patient with rheumatoid arthritis comprising a capture drug antibody and a tracer drug antibody, wherein

a) the capture drug antibody and the tracer drug antibody have a concentration of 0.5 μg/ml or more in the enzyme-linked immunosorbent assay,

b) incubating the sample simultaneously with the capture drug antibody and the tracer drug antibody for 0.5 to 24 hours,

c) the capture drug antibody is a 1:1 conjugate of the drug antibody and the first component of a specific binding pair, and the tracer drug antibody is a 1:1 conjugate of the drug antibody and a detectable label,

d) the sample contains at least 1% serum,

e) Oligomeric human IgG is added to the sample prior to incubation with the capture drug antibody and the tracer drug antibody.

In one embodiment, the sample comprises at least 5% serum. In one embodiment the sample comprises at least 7.5% serum.

In one embodiment, the sample comprises an anti-drug antibody and rheumatoid factor.

In one embodiment, the drug antibody is an antibody for the treatment of an inflammatory disease. In one embodiment, the antibody for the treatment of the inflammatory disease is an antibody for the treatment of an autoimmune disease. In one embodiment the autoimmune disease is rheumatoid arthritis or juvenile arthritis or osteoarthritis or Castleman's disease.

In one embodiment, the drug antibody is an antibody for the treatment of cancer. In one embodiment the antibody is for treatment of myeloma or plasmacytoma.

In one embodiment, the drug antibody is an antibody against IL-6 receptor (anti-IL6R antibody), or against IGF-1 receptor (anti-IGF1R antibody), or IL13 receptor 1 alpha (anti-IL13R1 alpha antibody), Or an antibody against Ox40L (anti-Ox40L antibody) or against tumor necrosis factor alpha (anti-TNFalpha antibody). In one embodiment the drug antibody is an anti-IL6R antibody. In one embodiment the anti-IL6R antibody is tocilizumab.

In one embodiment, the drug antibody is conjugated to a solid phase. In one embodiment, conjugation of the drug antibody to the solid phase is carried out through a specific binding pair. In one embodiment, the specific binding pair (first component/second component) is streptavidin or avidin/biotin, or an antibody/antigen (see, eg, Hermanson, GT, et al., Bioconjugate Techniques, Academic Press. , 1996), or lectin/polysaccharide, or steroid/steroid binding protein, or hormone/hormone receptor, or enzyme/substrate, or IgG/protein A and/or G.

In one embodiment, the drug antibody is conjugated to biotin (as the first component of a specific binding pair). In this case, the conjugation to the solid phase is performed through immobilized avidin or streptavidin.

In one embodiment, the drug antibody is conjugated to a detectable label. In one embodiment, the drug antibody is conjugated to the detectable label through a specific binding pair. In one embodiment, the specific binding pair (first component/second component) is streptavidin or avidin/biotin, or an antibody/antigen (see, eg, Hermanson, GT, et al., Bioconjugate Techniques, Academic Press. , 1996), or lectin/polysaccharide, or steroid/steroid binding protein, or hormone/hormone receptor, or enzyme/substrate, or IgG/protein A and/or G.

In one embodiment, the drug antibody is conjugated to digoxigenin (as a detectable label). In this case, binding to the detectable label is performed through an antibody against digoxigenin.

In one embodiment, the capture drug antibody and the tracer drug antibody have a concentration of about 0.5 μg/ml to about 10 μg/ml in the enzyme-linked immunosorbent assay (ELISA). In one embodiment the capture drug antibody and the tracer drug antibody have a concentration of greater than 0.5 μg/ml and less than 10 μg/ml. In one embodiment, the capture drug antibody and the tracer drug antibody have a concentration of about 1 μg/ml to about 5 μg/ml. In one embodiment, the capture drug antibody and the tracer drug antibody have a concentration of about 1.4 μg/ml to about 1.8 μg/ml. In one embodiment the capture drug antibody and the tracer drug antibody have a concentration of about 1.45 μg/ml to about 1.6 μg/ml. In one preferred embodiment, the capture drug antibody and the tracer drug antibody have a concentration of about 1.5 μg/ml.

In one embodiment the incubation time is at least 6 hours. In one embodiment the incubation time is at least 12 hours. In one embodiment the incubation time is at least 16 hours.

In one embodiment the incubation time is 24 hours or less.

In one embodiment, the incubation time is between 4 hours and 24 hours. In one embodiment, the incubation time is 6 to 24 hours. In one embodiment, the incubation time is 12 to 24 hours. In one embodiment the incubation time is 12 to 20 hours. In one embodiment the incubation time is 14 to 18 hours. In one embodiment the incubation time is about 16 hours.

In one embodiment the sample comprises 1% to 20% serum. In one embodiment the sample comprises about 10% serum.

In one embodiment the oligomeric human IgG is added at a final concentration of 10 μg/ml to 1000 μg/ml. In one embodiment the oligomeric human IgG is added at a final concentration of 15 μg/ml to 500 μg/ml. In one embodiment the oligomeric human IgG is added at a final concentration of 20 μg/ml to 250 μg/ml. In one embodiment the oligomeric human IgG is added at a final concentration of 25 μg/ml to 100 μg/ml. In one embodiment the oligomeric human IgG is added to a final concentration of about 50 μg/ml.

One aspect as reported in the present invention is an enzyme-linked immunosorbent assay for detection of an anti-drug antibody against a drug antibody in a sample of a patient with rheumatoid arthritis comprising a capture drug antibody and a tracer drug antibody, wherein

a) the capture drug antibody and the tracer drug antibody have a concentration of 1.5 μg/ml in the enzyme-linked immunosorbent assay,

b) incubating the sample simultaneously with the capture drug antibody and the tracer drug antibody for 14 to 16 hours,

c) The capture drug antibody is a 1:1 conjugate of the drug antibody and biotin through the lysine residue of the drug antibody, and the tracer drug antibody is 1:1 of the drug antibody and digoxigenin through the lysine residue of the drug antibody. Is a conjugate,

d) the sample contains 1% to 20% of serum,

e) Oligomeric human IgG is added to the sample at a final concentration of 25 μg/ml to 100 μg/ml prior to incubation with the capture drug antibody and the tracer drug antibody.

One aspect as reported herein is the use of oligomeric human IgG for the capture of rheumatoid factors in an anti-drug antibody ELISA.

One aspect as reported in the present invention comprises administering to a diseased individual an effective amount of a therapeutic antibody (drug) and measuring the presence of an anti-drug antibody by an assay as reported in the present invention. It's a treatment method.

One aspect as reported in the present invention is to administer an effective amount of an anti-IL6R antibody (drug) to an individual with an inflammatory disease and an anti-anti-IL6R antibody antibody (anti-drug antibody) with an assay as reported in the present invention. ) Is a method of treating the subject comprising measuring the presence of.

In one embodiment the inflammatory disease is an autoimmune disease. In one embodiment the autoimmune disease is selected from rheumatoid arthritis, juvenile arthritis, osteoarthritis, or Castleman's disease.

In one embodiment the inflammatory disease is intervascular proliferative glomerulonephritis.

One embodiment as reported in the present invention is to administer an effective amount of an anti-IL6R antibody (drug) to an individual with plasmacytoma and an anti-anti-IL6R antibody antibody (anti-drug antibody) by an assay as reported in the present invention. ) Is a method of treating the subject comprising measuring the presence of.

One aspect as reported in the present invention is to administer an effective amount of an anti-IL6R antibody (drug) to an individual with myeloma and an anti-anti-IL6R antibody antibody (anti-drug antibody) as reported in the present invention. It is a method of treating the subject comprising measuring the presence of.

One aspect as reported in the present invention is to administer to an individual an amount of an anti-IL6R antibody effective to inhibit IL6R activity, and an anti-anti-IL6R antibody antibody (anti-drug antibody) with an assay as reported in the present invention. ) Is a method of inhibiting IL6R activity in the subject comprising measuring the presence of.

1 is an analytical principle of an exemplified drug-resistant anti-drug antibody assay for anti-IL6R antibody tocilizumab.
Figure 2 is an analysis principle of the analysis of interference-suppressed anti-drug antibodies to anti-IL6R antibody tocilizumab.
3 is a calibration curve obtained for the analysis of interference-suppressed anti-drug antibodies to anti-IL6R antibody tocilizumab.
Figure 4 is a comparison of drug resistance of a two-step anti-drug antibody ELISA against anti-IL6R antibody tocilizumab and an interference-suppressed anti-drug antibody ELISA as reported herein; The signal of 300 ng/ml anti-drug antibody in the presence of increasing amounts of drug is shown; Dotted line: typical analysis CP; Solid line: Interference-suppressed assay CP as reported in the present invention; Dotted line with circles: conventional analysis; Solid line with squares: Interference-suppressed analysis as reported in the present invention.
5 is a cut-point measurement by interference-suppressed anti-drug antibody ELISA.
Figure 6 is a cut-point measurement by an interference-suppressed anti-drug antibody ELISA for TCZ-Bi (single) and TCZ-Dig (single).
7 is signal fluctuation using conventional ELISA in 77 different serum samples from TCZ-treated RA patients.
8 is a signal fluctuation using an interference suppressed ELISA as reported herein in 77 different serum samples from TCZ-treated RA patients.

Justice

The term “1:1 conjugate” refers to a conjugate consisting of exactly two entities bonded/conjugated to each other through a single covalent bond. For example, the term “a 1:1 conjugate of a drug antibody and a first component of a particular binding pair” refers to a drug antibody covalently conjugated to exactly one molecule of the first component of a particular binding pair through a single chemical bond. It represents a chemical conjugate consisting of exactly one molecule. Likewise, the term “a 1:1 conjugate of a drug antibody and a detectable label” refers to a chemical conjugate consisting of exactly one molecule of a drug antibody covalently conjugated to exactly one detectable label molecule through a single chemical bond.

The term "drug antibody" according to the present invention refers to an antibody that can be administered to a subject, and therefore, the sample of the subject is suspected to contain the drug antibody after administration. Drug antibodies are antibodies to be administered to humans for the purpose of treatment. Within one assay as reported herein, said drug antibody, said capture drug antibody and said tracer drug antibody are produced recombinantly by "same" antibody molecules, e.g., the same expression vector, and have the same amino acid sequence. It includes the containing antibody molecule. Drug antibodies (therapeutic monoclonal antibodies) are widely used in the treatment of various diseases, such as oncological diseases (e.g., hematologic and solid cancers, such as non-Hodgkin's lymphoma, breast cancer, and colorectal cancer) or inflammatory diseases. Is used. Such antibodies are described, for example, in Levene, A.P., et al., Journal of the Royal Society of Medicine 98 (2005) 145-152; Groner, B., et al., Curr. Mol. Meth. 4 (2004) 539-547]; And Harris, M., Lancet Oncol. 5 (2004) 292-302.

In one embodiment, the drug antibody is an antibody useful for the treatment of an inflammatory disease, i.e. an anti-inflammatory antibody, for example an anti-IL6 receptor antibody, or an anti-IGF-1 receptor antibody, or an anti-IL-13 receptor 1 alpha antibody. to be.

An exemplary (preferably monoclonal) drug antibody is an antibody against the IL-6 receptor (anti IL6R antibody). Such antibodies are described, for example, in Mihara, et al., Clin. Immunol. 98 (2001) 319-326]; Nishimoto, N., et al, Blood 106 (2005) 2627-2632, clinical trial NCT00046774, or WO 2004/096274.

An exemplary (preferably monoclonal) drug antibody is an antibody against the IGF-1 receptor (anti IGF1R antibody). Such antibodies are reported for example in WO 2004/087756 or WO 2005/005635.

An exemplary (preferably monoclonal) drug antibody is an antibody against IL-13 receptor alpha (anti IL13R1 alpha antibody). Antibodies against IL-13R1 alpha are described, for example, in WO 　 96/29417, WO 　 97/15663, WO 　 03/080675, Graber, P., et al., Eur. J. Immunol. 28 (1998) 4286-4298]; See Poudrier, J., et al., J. Immunol. 163 (1999) 1153-1161]; See Poudrier, J., et al., Eur. J. Immunol. 30 (2000) 3157-3164]; It is known from Aikawa, M., et al., Cytokine 13 (2001) 75-84, and is commercially available, for example from R&D Systems Inc. (USA). have. Additional exemplary antibodies against IL-13R1alpha are reported in WO 2006/072564.

The term "drug antibody used in anti-inflammatory therapy" as used herein refers to a drug antibody against a cell surface receptor that mediates inflammation. Such a receptor is, for example, the IL-6 receptor, or the IGF-1 receptor, or the IL-13a receptor 1. When analyzing a sample from a patient treated with an anti-inflammatory drug antibody as described above, whether the positive result of the method is based on a true anti-drug antibody (true positive result) or an antibody other than the anti-drug antibody of the sample ( False positive results). An example of such a case is a sample from a patient with an autoimmune disease such as rheumatism, and thus a sample obtained from the patient contains a so-called "rheumatic factor". As used herein, the term "rheumatic factor" refers to an antibody that binds to human IgG, more strictly to the Fc-region of human IgG. In most cases the "rheumatic factor" is an oligomeric binding molecule.

The term "anti-drug antibody" as used herein refers to an antibody that binds to, ie, an antigenic region of a drug antibody. The antigenic region may be a variable region, a CDR, a constant region, or a sugar structure of the drug antibody. In one embodiment, the anti-drug antibody is produced from the recombinant production of the drug antibody against the CDR of the drug antibody, or in a recombinant cell, e.g., CHO cell, HEK cell, Sp2/0 cell, or BHK cell. It is a secondary modification of the drug antibody. In general, anti-drug antibodies are directed against the antigenic region of the drug antibody that is recognized by the immune system of the animal to which the drug antibody is administered. The antibodies disclosed above are referred to as "specific anti-drug antibodies".

Drug antibodies are designed to contain as few antigenic regions as possible. For example, a drug antibody intended for use in humans is humanized prior to application to a human patient in order to minimize the occurrence of an immune response to the drug antibody. The immune response may be in the form of an anti-drug antibody (ADA) against a non-human portion of such a humanized drug antibody, for example, a complementarity determining region of the variable domain (see, for example, Pan, Y ., et al., FASEB J. 9 (1995) 43-49).

The term "hypervariable region" or "HVR" as used herein means that the sequence is highly variable ("complementary determining region" or "CDR") and/or forms a structurally defined ring ("hypervariable ring"), and Refers to the respective regions of an antibody variable domain containing an antigen-contacting moiety (“antigen contact”) In general, the antibody is of 6 HVRs, ie 3 of VH (H1, H2, H3) and VL. 3 (L1, L2, L3). An exemplary HVR in the present invention is

(a) present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) Hypervariable rings (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3). CDR (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

(c) present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3). Antigen contacts (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); And

(d) HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49- Combinations of (a), (b) and/or (c), including 65 (H2), 93-102 (H3), and 94-102 (H3)

Includes.

Unless otherwise indicated, HVR residues and other residues (eg FR residues) in the variable domain are numbered according to Kabat in the present invention.

Antibodies contain a number of reactive moieties, such as amino groups (lysine, alpha-amino groups), thiol groups (cystine, cysteine and methionine), carboxylic acid groups (aspartic acid, glutamic acid) and sugar-alcohol groups. They can be used to couple to a binding partner such as a surface, a protein, a polymer (eg PEG, cellulose or polystyrol), an enzyme or a member of a binding pair (see, eg Aslam M. and Dent, A. , Bioconjuation MacMillan Ref. Ltd. (1999) 50-100).

The term “anti-genotype antibody” refers to an antibody that specifically binds to the binding specificity of the parent antibody, eg, a binding site, ie an anti-genotype antibody is directed to, eg, an antigen binding site of the parent antibody.

In one embodiment the anti-genotype antibody specifically binds to one or more of the CDRs of the parent antibody.

In one embodiment the parent antibody is a therapeutic antibody. In one embodiment the parent antibody is a multispecific antibody. In one embodiment the parent antibody is a bispecific antibody.

One of the most common reactive groups for proteins is the aliphatic ε-amine of the amino acid lysine. In general, almost all antibodies contain abundant lysine residues. The lysine amine/amino group is a fairly good nucleophile (pK _a = 9.18) above pH 8.0 and thus reacts easily and cleanly with various reagents to form stable bonds.

Another common reactive group in antibodies is the thiol residue from the sulfur-containing amino acid cystine and its reduction product, cysteine (or half cystine). Cysteine contains free thiol groups, which are more nucleophilic than amines and are generally the most reactive functional groups in proteins. Thiols are generally reactive at neutral pH and thus can optionally couple to other molecules in the presence of an amine. Since the free sulfhydryl group is relatively reactive, proteins bearing this group are often present with the groups in oxidized form as disulfide groups or disulfide bonds.

Besides cystine and cysteine, some proteins also have the amino acid methionine, which contains sulfur in the thioether bond. The literature describes a number of thiolated crosslinking reagents, such as Traut's reagent (2-iminothiolane), succinimidyl (acetyl), to provide an efficient way to introduce multiple sulfhydryl groups via reactive amino groups. The use of thio) acetate (SATA), or sulfosuccinimidyl 6-[3-(2-pyridyldithio) propionamido] hexanoate (sulfo-LC-SPDP) is reported.

Reactive esters, in particular N-hydroxysuccinimide (NHS) esters, are one of the most commonly used reagents for the modification of amine groups. The optimum pH for the reaction in an aqueous environment is between pH 8.0 and 9.0.

Isothiocyanates are amine-modifying reagents and form thiourea bonds with proteins. It reacts with protein amines in aqueous solution (optimally at pH 9.0 to 9.5).

Aldehydes react with aliphatic and aromatic amines, hydrazines and hydrazides under mild aqueous conditions to form imine intermediates (Schiff's base). Stable alkyl amine bonds can be induced by selectively reducing Schiff's base with a mild or strong reducing agent (eg sodium borohydride or sodium cyanoborohydride).

Another reagent used to modify the amine is acid anhydride. For example, diethylenetriaminepentaacetic anhydride (DTPA) is a difunctional chelating agent containing two amine-reactive anhydride groups. This can be reacted with the N-terminus of the protein and the ε-amine group to form an amide bond. The anhydride ring is ring-opened to produce a multivalent metal-chelated branch capable of tightly binding to the metal in the coordination complex.

The term “sample” includes, but is not limited to, any amount of material from alive or previously alive. Such living things include, but are not limited to, humans, mice, monkeys, rats, rabbits and other animals. In one embodiment the sample is obtained from a monkey, in particular a cynomolgus monkey, or a rabbit, or a mouse or rat. Such substances include, but are not limited to, whole blood, serum or plasma from individuals, which in one embodiment are the most widely used sample sources for clinical pathways.

The term "solid phase" refers to a non-fluid phase material, including particles (including microparticles and beads) made from materials such as polymers, metals (paramagnetic, ferromagnetic particles), glass and ceramics; Gel materials such as silica, alumina, and polymer gels; Capillaries, which may be made of polymers, metals, glass and/or ceramics; Zeolites and other porous materials; electrode; Microtiter plate; Solid strip; And cuvettes, tubes or other spectrometer sample containers. Solid phase components are distinguished from inert solid surfaces in that the "solid phase" contains, on its surface, one or more portions that are intended to interact with a substance in the sample. The solid phase may be a stationary phase component, such as a tube, strip, cuvette or microtiter plate, or a non-stationary phase component, such as beads and microparticles. A variety of microparticles can be used that allow for both non-covalent or covalent binding of proteins and other substances. Such particles include polymer particles such as polystyrene and poly(methylmethacrylate); Gold particles such as gold nanoparticles and gold colloids; And ceramic particles such as silica, glass and metal oxide particles. See, for example, Martin, C.R., et al., Analytical Chemistry-News & Features, 70 (1998) 322A-327A, or Butler, J.E., Methods 22 (2000) 4-23.

In one embodiment, a detectable label is selected from chromogens (fluorescent or luminescent groups and dyes), enzymes, NMR-activators, metal particles, or haptens, such as digoxigenin. In one embodiment the detectable label is digoxigenin. The detectable label may also be a photoactivatable crosslinking group, such as an azido or azirine group. Metal chelates that can be detected by electrochemiluminescence are also signal-emitters in one embodiment, with ruthenium chelates, for example ruthenium(bispyridyl) ₃ ²⁺ chelates being particularly preferred. Suitable ruthenium labeling groups are disclosed, for example, in EP 0 580 979, WO 90/05301, WO 90/11511, and WO 92/14138.

The principles of different immunoassays are described, for example, in Hage, D.S., Anal. Chem. 71 (1999) 294R-304R. Lu, B., et al., Analyst 121 (1996) 29R-32R reports oriented immobilization of antibodies for use in immunoassays. Avidin-biotin-mediated immunoassays are described, for example, in Wilchek, M., and Bayer, E.A., Methods Enzymol. 184 (1990) 467-469.

Detailed description of the invention

The present invention reports an interference-suppressed anti-drug antibody assay using serum samples with increased resistance to free therapeutic antibodies and increased resistance to rheumatoid factor interference.

The principle of the anti-drug antibody assay is a complex with a digoxigenylated drug (drug-Dig) and a biotinylated drug (drug-Bi) (e.g. tocilizumab (TCZ-Dig and TCZ-Bi, respectively)). Is the capture of anti-drug antibody (ADA), and the drug-Bi is immobilized on a streptavidin-coated plate (SA-MTP). The ADA/drug-Dig complex bound to the drug-Bi on the SA-MTP is detected by the anti-digoxigenin antibody horseradish peroxidase enzyme conjugate (anti-Dig-HRP). The horseradish peroxidase (HRP) catalyzes the color reaction of the substrate ABTS. The color intensity is proportional to the concentration of the analyte. The general principle of anti-drug antibody assay is shown in FIG. 1.

Drug and rheumatoid factor resistance in conventional anti-drug antibody assays without changing the general analytical principles.

1) increasing the concentration of biotinylated and digoxigenylated capture and tracking reagents;

2) simultaneous cultivation of the capture and tracking reagent and the serum sample instead of continuous cultivation;

3) extended incubation time between the serum sample and the capture and tracking reagent;

4) use of homogeneous capture and tracking reagents instead of heterogeneous coupled mixtures;

5) the use of increased serum substrates;

6) the inclusion of oligomeric IgG as an analytical additive; And

7) Use of single biotinylated capture and single digoxigenylated tracer antibody

Can be increased by

These means provided a synergistic effect.

These measures lead to an interference-suppressed drug-resistant anti-drug antibody assay for detection of anti-drug antibodies against therapeutic drug antibodies.

The general principle of the interference-suppressed drug-resistant anti-drug antibody assay as reported herein is shown in Figure 2 illustrated for the anti-IL6R antibody tocilizumab.

By the assay configuration as reported in the present invention, the drug resistance of the ADA assay was increased by more than 10 times in serum samples from patients compared to conventional anti-drug antibody assays. At the same time, the sensitivity to rheumatic factors leading to false positive analysis was also reduced.

The therapeutic anti-inflammatory antibody tocilizumab (TCZ) is a recombinant humanized monoclonal antibody against the interleukin-6 receptor. It has been shown to be effective in clinical studies of rheumatoid arthritis (Ohsugi, Y. and Kishimoto, T., Expert Opin. Biol. Ther. 8 (2008) 669-681). The ADA screening and confirmation assays used in this study showed sufficient drug resistance to typical TCZ serum concentrations reached steady state using an intravenous dosing regimen.

However, different routes of administration, such as subcutaneous administration, more frequent administration, and new indications in children may result in higher TCZ serum concentrations under normal conditions.

In addition, for example, rheumatoid factor (RF) is often significantly increased in patients with autoimmune diseases, such as rheumatoid arthritis. RF exhibits dominant binding to aggregated gamma globulin and is involved in the mechanism of elimination of immune complexes in vivo (Tatarewicz, S., et al., J. Immunol. Methods. 357 (2010) 10-16)). . RF is predominantly pentameric immunoglobulin M (IgM) isotype (Artandi, SE, et al., Proc. Natl. Acad. Sci. USA 89 (1991) 94-98) and false positive results in ADA analysis. It can nonspecifically bind with a multivalent intermediate affinity to the constant portion of the therapeutic antibody that induces. For example, affinity purified rabbit anti-human IgM antibody was included in the sample diluent to overcome cross-reactive IgM antibody interference in RA samples (see, eg, Araujo, J., et al., J. Pharm. Biomed. Anal., 55 (2011) 1041-1049).

It has been found that non-specific binding of RF present in the serum sample to the therapeutic antibody can be prevented by adding oligomeric human IgG to the sample prior to performing the ADA assay. The added oligomeric IgG provides an additional target for the RF and at best provides interference of RF in ADA assays as reported herein.

In the following the interference-suppressed ADA assay as reported herein is illustrated by analysis of serum samples from patients with tocilizumab (TCZ) treated rheumatoid arthritis.

Means for increasing drug resistance of conventional anti-drug antibody assays for detecting anti-drug antibodies against anti-IL6R antibody tocilizumab are:

1) increasing the concentration of biotinylated and digoxigenylated TCZ (eg 0.5 μg/ml to 1.5 μg/ml);

2) incubating the serum samples simultaneously with TCZ-Bi and TCZ-Dig;

3) prolonged incubation of the serum sample with TCZ-Bi and TCZ-Dig (eg 1 to 16 hours);

4) Instead of a mixture of lysine- and carbohydrate-coupled reagents, only lysine-coupled TCZ-Bi and TCZ-Dig reagents are used;

5) use increased serum substrate content;

6) Oligomeric human IgG was added to the sample before incubation of TCZ-Bi and TCZ-Dig

Was.

Drug resistance

The concentration of anti-IL6R antibody tocilizumab detected in clinical samples is at least 0.5 μg/ml, often in the range of 1 μg/ml to 10 μg/ml.

The drug resistance of the conventional anti-drug antibody assay was evaluated by measuring the highest TCZ concentration that a given concentration of positive control ADA could be detected above the cut-point. Table 1 presents a summary of the results.

[Table 1]

An ADA concentration of 125 ng/ml was detected and tested positive in the presence of 10 μg/ml TCZ.

The drug resistance of the interference-suppressed drug-resistant anti-drug antibody assay as reported in the present invention was evaluated by measuring the highest TCZ concentration that a given concentration of positive control ADA could be detected above the cut-point. . Table 2a presents a summary of the results.

[Table 2a]

A very low ADA concentration of 30 ng/ml was detected in the interference-suppressed drug-resistant anti-drug antibody ELISA as reported in the present invention and tested positive in the presence of 10 μg/ml TCZ. Moreover, the ADA concentrations of 100 ng/ml and 300 ng/ml respectively show drug antibody resistance of 30 μg/ml and even 100 μg/ml.

The interference-suppressed assay revealed a 10-fold higher drug resistance in comparison to the same experiment performed by the previously used two-step conventional anti-drug antibody ELISA against tocilizumab (300 ng/ See also Figure 4 for ml ADA concentration).

Drug resistance of the interference-suppressed drug-resistant anti-drug antibody assay as reported in the present invention for a 1:1 conjugate of biotin and digoxigenin monovalently bound to a drug antibody, respectively, for a given concentration of positive control ADA was evaluated by measuring the highest TCZ concentration that could be detected above the cut-point. Table 3 presents a summary of the results.

[Table 3]

In the interference-suppressed drug-resistant anti-drug antibody ELISA as reported in the present invention, an ADA concentration of 250 ng/ml was detected and tested positive in the presence of 80 μg/ml TCZ.

Interference suppression:

Sixteen clinical serum samples were analyzed by conventional anti-drug antibody assays as disclosed in Stubenrauch et al., supra. The results are summarized in Table 4a.

[Table 4a]

Without changing the analysis principle, a series of measurements were performed to obtain an interference-suppressed drug-resistant anti-drug antibody ELISA as reported in the present invention.

Above

1) increasing the concentration of biotinylated and digoxigenylated TCZ (eg 0.5 μg/ml to 1.5 μg/ml);

2) incubating the serum samples simultaneously with TCZ-Bi and TCZ-Dig;

3) prolonged incubation of the serum sample with TCZ-Bi and TCZ-Dig (eg 1 to 16 hours);

4) Instead of a mixture of lysine- and carbohydrate-coupled reagents, only lysine-coupled TCZ-Bi and TCZ-Dig reagents are used;

5) use increased serum substrate content;

6) Oligomeric human IgG was added to the sample before incubation of TCZ-Bi and TCZ-Dig

to be.

The results as obtained by the interference suppressed assay as reported in the present invention are shown in Table 4b below.

[Table 4b]

It has been found that when only some of the measures outlined above are performed, the reduction in interference is not sufficient and sensitivity to interference by existing rheumatic factors still produces false positive ADA analysis results.

For example just

1) increasing the concentration of biotinylated and digoxigenylated TCZ (eg 0.5 μg/ml to 1.5 μg/ml);

2) incubating the serum samples simultaneously with TCZ-Bi and TCZ-Dig;

3) prolonged incubation of the serum sample with TCZ-Bi and TCZ-Dig (eg 1 to 16 hours);

4) Instead of a mixture of lysine- and carbohydrate-coupled reagents, only lysine-coupled TCZ-Bi and TCZ-Dig reagents are used;

5) using increased serum substrate content

In the case of performing only the means, a sufficient decrease in sensitivity to false-positive assay results cannot be observed. Comparative data are shown in Table 4c.

[Table 4c]

Comparative evaluation of 258 different serum samples from TCZ-treated RA patients by conventional ADA assay and interference-suppressed drug resistance ADA assay as reported herein showed the same positive results in 12 samples. The conventional assay determined that 27 placebo patients were positive, whereas the interference-suppressed drug resistance ADA assay as reported in the present invention was only 4 patients. In conclusion, the combination of the means as disclosed herein and the addition of oligomeric human IgG as an ADA assay additive conferred increased drug resistance and interference by suppressed RF compared to conventional ADA assays.

A subset analysis of the data mentioned above is shown in Table 7a below.

[Table 7a]

Study-related CP: 0.215 (conventional ELISA); 0.058 (interference-suppressed ELISA); +: positive ELISA result; -: negative ELISA result

Analysis signals for 27 placebo patients not treated with TCZ in Table 8aa are shown. Due to the absence of TCZ-treatment induced ADA, a high assay signal in this group is not expected and the signal will indicate potential interference.

[Table 8aa]

Study-related cut-point (CP): 0.215 (conventional ELISA); 0.058 (interference-suppressed ELISA); +: positive ELISA result; -: negative ELISA result

The signal patterns of both assays are very different: all 27 placebo samples were determined to be positive using conventional ELISA, whereas only 4 of the 27 samples for the interference-suppressed ELISA as reported in the present invention. Only dogs were determined to be positive.

In addition to placebo-treated patients, samples from TCZ-treated patients were analyzed. Table 8b shows the results for patients before TCZ-treatment. Results for TCZ-treated patients are shown in Table 8c.

[Table 8b]

Study-related CP: 0.215 (conventional ELISA); 0.058 (interference-suppressed ELISA); +: positive ELISA result; -: negative ELISA result

[Table 8c]

Study-related CP: 0.215 (conventional ELISA); 0.058 (interference-suppressed ELISA); +: positive ELISA result; -: negative ELISA result

Assays as reported herein provide advantages that are independent of the therapeutic antibody and target used. This is shown in the following table for anti-IL6R antibody, anti-IGF-1R antibody, anti-IL13R alpha antibody, anti-OX40L antibody and anti-Abeta antibody using samples from patients diagnosed as positive for rheumatoid arthritis. .

[Table 9a]

[Table 9b]

[Table 9c]

[Table 9d]

[Table 9e]

The following examples and drawings are provided to aid in understanding the present invention, the true scope of which is set forth in the appended claims. It is believed that the modification can be carried out in the process described without departing from the true meaning of the present invention.

Example

Substance and method

Purified pooled human immunoglobulin class G (IgG) was described in Stubenrauch et al., Anal. Biochem. 390 (2009) 189-196. Briefly, human pooled serum from healthy donors was delipidated with Aerosil (silicon dioxide, 1.5% (w/v)) and precipitated with ammonium sulfate (ad 2.0 M). The pellet was homogenized in phosphate buffer and dialyzed against phosphate buffer (pH 7.0).

The mixture was separated by DEAE ion exchange chromatography at pH 7.0, and the IgG in the treatment solution was concentrated to 5.93 mg/ml and purified by gel filtration.

Polyclonal anti-digoxigenin-horseradish peroxidase (HRP) conjugate (Fab fragment) was obtained from Roche Diagnostics GmbH (Mannheim, Germany) (cat.no. 11633716). Polyclonal rabbit anti-TCZ antibody (0.5 mg-equivalent/ml) used as positive quality control (QC) and calibration standard (CS) was prepared as disclosed in Stubenrauch et al., supra.

Individual human serum samples were provided by the Serum Bank of Roche Diagnostics GmbH (Pennsberg, Germany). The human pooled serum substrate for negative control was supplied by TCS Biosciences Ltd. (Buckingham, UK).

The following reagents were obtained from Roche Diagnostics GmbH (Mannheim, Germany): 2,2'-azido-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) substrate (cat.no. 11684302-) 001), washing buffer for ELISA: phosphate-buffered saline (PBS) (0.01M KH2PO4, 0.1M Na2HPO4, 1.37M NaCl, 0.027M KCl; pH 7.0)/0.05% polysorbate 20 (twin 20) (cat. no.11332465-001), and a ready-to-use universal buffer used as a dilution buffer in ELISA (cat no.4742672).

Streptavidin-coated microtiter plates (SA-MTP) were obtained from MicroCoat Biotechnologie GmbH (Bernreed, Germany). Uncoated Nunc 96-microwell plates were obtained from Fisher Scientific GmbH (Schubert, Germany) (cat no. 442587) and used for pre-culture.

Conventional anti-drug antibody assay:

The analysis was carried out at room temperature. In the first step, TCZ-Bi was bound to SA-MTP at a concentration of 0.5 μg/ml by incubating 100 μl for 1 hour at 400 rpm on a shaker. Before adding the pre-culture solution to the SA-MTP, excess unbound TCZ-Bi was removed by washing 3 times. In parallel with the above coating process, pre-culture of standards and samples was performed in duplicate in separate uncoated 96-well plates. The samples and standards were diluted to a volume of 75 μl in wells with 10% serum substrate (1:10) and mixed with an equal volume of TCZ-DIG to begin a 1 hour pre-incubation period. The TCZ-Bi-coated SA-MTP was loaded with the pre-culture solution by transferring 100 µl from each well of the pre-culture plate to the well of the coated MTP, and 1 hour at 400 rpm on a shaker. During incubation. After washing, polyclonal anti-Dig horseradish peroxidase (HRP) conjugate in a volume of 100 μl (100 mU/ml) was added to the wells and incubated for 1 hour on a shaker. After washing, the HRP-catalyzed color-generating reaction was initiated by the addition of 100 μl ABTS solution. When the maximum optical density (OD) was about 2.0, usually within 20 to 30 minutes, the signal of the color response was measured by an ELISA reader at a wavelength of 405 nm (reference, 490 nm). The same assay was carried out in the presence of a confirming reagent, and at the same time measurements were performed without the confirming reagent. The obtained OD data was used to generate a standard calibration curve by nonlinear four-parameter regression curve matching according to the Wiemer-Rodbard method for calculation of the sample concentration.

The cutoff points of the test results were assigned at 95% CI of the assay signal (OD) from multiple analyzes of human blank serum samples from healthy volunteers and RA patients. Screening test results were considered positive at values above the cutoff. A reduction in absorbance of >20% compared to the non-spiked sample indicated a positive result. The selection cutoff was measured at 61.4 ng/ml of the reference antibody. The intra- and inter-analytical accuracy was 84.8% to 93.1% and 91.3% to 92.2%, respectively. Corresponding values for the intra- and inter-analytical precision were 1.8% to 2.0% and 6.8% to 8.0%. The accuracy of the ELISA was defined by the degree to which the analysis result was consistent with the true value. Accuracy was determined by comparing the measured concentration of rabbit polyclonal anti-TCZ-positive control standard spiked in human serum to the nominal concentration of anti-TCZ. High (360 ng equivalents/ml) and low (60 ng equivalents/ml) positive control, 6 aliquots of each positive control measured in duplicate to determine intra-assay accuracy, and inter-analytical accuracy. To measure, it was analyzed in three aliquots of each positive control, measured in duplicate.

Example One

Biotinylation of anti-IL6R antibody tocilizumab

a) Preparation of conventionally biotinylated IgG

The anti-IL6R antibody tocilizumab was dialyzed against buffer (100 mM potassium phosphate buffer (referred to below as K-PO4), pH 8.5). Thereafter, the solution was adjusted to a protein concentration of 5 mg/ml. D-biotinoyl-aminocaproic acid-N-hydroxysuccinimide ester was dissolved in dimethyl sulfoxide (DMSO) and added to the antibody solution at a molar ratio of 1:5. After 60 minutes the reaction was stopped by addition of L-lysine. Excess of the labeling reagent was removed by dialysis against 50 mM K-PO4 supplemented with 150 mM KCl, pH 7.5. An aliquot of TCZ-Bi was stored including 6.5% sucrose at -80°C.

b) Preparation of single biotinylated IgG

The anti-IL6R antibody tocilizumab was dialyzed against 100 mM K-PO4, pH 8.5, after which the solution was adjusted to a protein concentration of 5 mg/ml. D-biotinoyl-aminocaproic acid-N-hydroxysuccinimide ester was dissolved in dimethyl sulfoxide (DMSO) and added to the antibody solution at a molar ratio of 1:1. After 60 minutes the reaction was stopped by addition of L-lysine. Excess of the labeling reagent was removed by dialysis against 50 mM K-PO4 supplemented with 150 mM KCl, pH 7.5. The mixture was transferred to a buffer with 100 mM K-PO4, 150 mM KCl, pH 7.2, containing 1M ammonium sulfate and applied to a column with streptavidin mutein Sepharose. Non-biotinylated IgG is in the treatment solution, single biotinylated IgG eluted with 100 mM K-PO4, 150 mM KCl, 1.5% DMSO, pH 7.2, and biotinylated containing a higher biotinylated population. IgG is eluted with 100 mM K-PO4, 150 mM KCl, 2 mM D-biotin, pH 7.2. The single biotinylated antibody was dialyzed against 50 mM K-PO4 supplemented with 150 mM KCl, pH 7.5. The aliquot was stored at -80 °C with 6.5% sucrose.

Example 2

Digoxigenylation of anti-IL6R antibody Tocilizumab

a) Preparation of conventionally digoxigenylated IgG

The anti-IL6R antibody tocilizumab was dialyzed against buffer (100 mM potassium phosphate buffer (referred to below as K-PO4), pH 8.5). Thereafter, the solution was adjusted to a protein concentration of 5 mg/ml. Digoxigenin 3-O-methylcarbonyl-ε-aminocaproic acid-N-hydroxysuccinimide ester was dissolved in DMSO and added to the antibody solution at a molar ratio of 1:4. After 60 minutes the reaction was stopped by addition of L-lysine. Excess of the labeling reagent was removed by dialysis against 50 mM K-PO4 supplemented with 150 mM KCl, pH 7.5. Digoxigenylated TCZ (TCZ-Dig) was stored in aliquots containing 6.5% sucrose at -80°C.

b) Preparation of single digoxigenylated IgG

The anti-IL6R antibody tocilizumab was dialyzed against 100 mM K-PO4, pH 8.5, after which the solution was adjusted to a protein concentration of 5 mg/ml. Digoxigenin 3-O-methylcarbonyl-ε-aminocaproic acid-N-hydroxysuccinimide ester was dissolved in dimethyl sulfoxide (DMSO) and added to the antibody solution at a molar ratio of 1:1. After 60 minutes the reaction was stopped by addition of L-lysine. Excess of the labeling reagent was removed by dialysis against 50 mM K-PO4 supplemented with 150 mM KCl, pH 7.5. The mixture was applied to a Sepharose column with an immobilized monoclonal antibody against digoxigenin. The non-digoxigenylated antibody was in the treatment solution, and the single digoxigenylated IgG was eluted with a mild elution buffer (Thermo Scientific, #21013), and the digoxigenylated population containing a higher digoxigenylated population. The genified antibody is eluted with 1M propionic acid. The fractions having the single digoxigenylated antibody were dialyzed first against 20 mM Tris, 20 mM NaCl, pH 7.5 and secondly against 50 mM K-PO4, 150 mM KCl, pH 7.5. The aliquots were stored at -80 °C with 6.5% sucrose.

Example 3

Generation of oligomer form human IgG

Human IgG purified from human serum by ion exchange chromatography was dialyzed against 150 mM potassium phosphate buffer containing 100 mM NaCl, pH 8.4, and the protein solution was concentrated to a protein concentration of 50 mg/ml. Disuccinimidyl suberate (DSS) was dissolved in DMSO and added to the antibody solution at a molar ratio of 1:6 (IgG:DSS). The mixture was incubated with stirring at 25° C. and pH 8.4 and the reaction was analyzed with an analytical gel filtration column (using a TSK 4000 column, for example). The polymerization was stopped by adding lysine to a final concentration of 20 mM after 140 minutes. After incubation at 25° C. for 45 minutes, the oligomeric human IgG was separated by gel filtration (for example, using a Sephacryl S400 column) to remove the low molecular weight fraction. The composition of the oligomer was characterized by UV spectroscopy, size exclusion chromatography and SDS-PAGE gel electrophoresis. Aliquot the oligomeric human IgG (10.5 mg/ml) and dilute it freshly with universal buffer (cat no. 4742672) at a concentration of 55.6 μg/ml for use as an ADA assay additive (AAA) in the immunoassay. Stored at -65 °C.

Example 4

Preparation of calibration standards and quality control samples

Mother liquors for calibration standards (CS) and quality control samples (QC) were prepared separately. The CS samples were prepared fresh on the day of analysis using 0.5 mg/ml stock solution of TCZ. After pre-dilution with human pooled serum (HPS), the resulting CS working solution was diluted 1:1 with 100% HPS stepwise before use in the assay to 1,000; 500; 250; 125; 62.5; 31.3; And a calibration concentration of 15.6 ng/ml. The negative control was 100% HPS. For the pre-culture step in the assay, the CS samples were diluted 1:10 to adjust a serum concentration of 10% and an assay concentration range of 100 ng/ml to 1.56 ng/ml.

The QC mother liquor samples used above were prepared from 100% human pooled serum and stored as a one-time aliquot at -20°C. Three separate QC samples were prepared and stored at mother liquor concentrations representing high (750 ng/ml), medium (400 ng/ml) and low (50 ng/ml) undiluted serum concentrations. For the pre-cultivation step in the assay, the QC samples were freshly diluted 1:10 in the capture/detection solution to reach a serum concentration of 10%. A fourth QC sample was further used at the typical cut-point of the above assay of 25 ng/ml.

Example 5

ADA screening and validation analysis

Sandwich ELISA was used for both screening and identification of anti-drug antibodies (ADA) against tocilizumab (TCZ) (Stubenrauch, K., et al., Clin. Ther. 32 (2010) 1597-1609). Please refer to). The principle of the method is the capture of ADA in a complex with TCZ-Dig and TCZ-Bi, which TCZ-Bi induces immobilization on streptavidin-coated plates. The TCZ-Bi/ADA/TCZ-Dig complex bound to the SA-MTP was detected by the anti-Dig-HRP enzyme conjugated antibody. The principle of the drug-resistant anti-drug antibody assay is shown in FIG. 1. The inclusion of oligomeric IgG as an ADA assay additive results in an interference-suppressed anti-drug antibody assay as shown in FIG. 2. Horseradish peroxidase (HRP) of the polyclonal antibody catalyzes the color reaction of the substrate ABTS. The color intensity is proportional to the concentration of the analyte.

The screening assay was performed at room temperature. Reagents and serum samples were diluted with universal buffer (cat no.4742672), and all washing steps were diluted with washing buffer (PBS, 0.05% Polysorbate 20 (Twin® 20) (cat.no. 11332465-001))). As, it was carried out three times at 300 µl per well. Incubation was carried out under shaking on a microtiter plate shaker (MTP shaker) at 500 rpm. Test samples, QC and CS samples were incubated overnight (up to 16 hours) with capture antibody TCZ-Bi and detection antibody TCZ-Dig. 225 μl of a capture/detection solution containing 1.667 μg/ml TCZ-Bi and 1.667 μg/ml TCZ-Dig with oligomeric human IgG ADA assay additive (AAA) in each well of the pre-culture microtiter plate (MTP) Was loaded and then 25 μl of each sample was added. The production concentrations of TCZ-Bi and TCZ-Dig were 1.5 µg/ml, respectively, and the oligomeric human IgG had a concentration of 50 µg/ml. The loaded MTP was covered to prevent evaporation and incubated overnight. 100 [mu]l of each well of the pre-culture plate was transferred to the wells of a streptavidin-coated microtiter plate (SA-MTP), covered and incubated for 1 hour.

After washing, the polyclonal anti-Dig Fab-HRP conjugate having a concentration of 25 mU/ml was added to each well in a volume of 100 μl and incubated for 1 hour. After washing, the ABTS ready-to-use solution was added to each well in 100 μl aliquots and incubated with shaking for about 10 to 15 minutes. The signal of the color-generating reaction was measured by an ELISA reader at a wavelength of 405 nm (reference wavelength: 490 nm). The absorbance value of each serum sample was measured in duplicate 3 times. The highest standard should reach an optical density (OD) in any unit (AU) of 1.8 to 2.2. The obtained OD data was used to generate a standard calibration curve by non-linear four-parameter matched “Bimmer Rodbart” for calculation of the sample concentration. The sample was found to be positive for ADA if the recovery of this concentration was below the specificity cut-point.

The assessment of this specificity cut-point was performed by analysis of individual blank human serum samples from 32 rheumatoid arthritis patients in replicates on one MTP. The cut-point specifies a signal above the signal that the sample is defined as being potentially positive for the presence of ADA in the ADA screening assay. Due to the non-normality of the data, a non-parametric approach with 95% percentile was applied to the cut-point calculation based on the average of the cut-points on the repeated plate.

The experiment performed for the cut-point determination of the ADA assay from duplicate measurements of individual human blank serum samples of 32 rheumatoid arthritis patients was a mean AU of about 0.026 on three different plates with a standard deviation (SD) of about 0.009. Revealed. The corresponding coefficients of variation (CV) are each 23.8%; 20.0%; And 19.0%. Based on these data sets, a normalization factor of NF = 1.6905 was derived and used throughout the assay qualitative analysis and plate-specific cut-point (plate-specific cut-point [AU] = signal [AU] (negative control). x NF).

For the qualitative analysis of the selective ADA assay, 7 calibration samples with an assay concentration range of 1.56 ng/ml to 100 ng/ml and 5 independent calibration curve formulations with blank samples were measured in duplicate on one plate. . The intra-analytical qualitative analysis run was performed in duplicate (5 separate vials), at which time 4 QC samples were each measured in duplicate on a single plate. Inter-analytical qualitative data for all QC samples measured in duplicate were obtained from 7 independent test runs performed by 2 or more operators on 4 different days.

A typical calibration curve of the interference-suppressed ELISA is shown in FIG. 3. The precision of duplicate measurements of the samples was evaluated during the qualitative analysis experiment and its CV did not exceed 15%. The intra- and inter-analytical precision and accuracy values of the interference-suppressed ADA ELISA are summarized in Table 5.

[Table 5]

The measured intra-analytical precision was <5% for all QCs including 25 ng/ml cut-point QC. The measured intra-analytical accuracy ranged from 91.1% to 102% and all cut-point QC samples tested positive. The inter-analytical precision for the inverse-calculated QC was <6% for all QCs. The measured inter-analytical accuracy was 92.8% to 105% for high, medium and low QC. All cut-point QC measurements gave ADA-positive.

Potential high-dose hook effects were assessed by serial titration (1:2) of positive control samples within an assay concentration range of 25,000 ng/ml to 6.1 ng/ml. The recovery of ADA concentration within the assay range was 77.9% to 98.9%. For analysis of potential substrate effects, 11 individual normal human serum samples were spiked and quantified with high-dose and low-dose QC, i.e. 50 and 750 ng/ml in 100% serum, with a positive control ADA. In addition, QC samples were also analyzed on the same plate. The recovery of the low and high ADA concentrations were 111% (range: 104-117%) and 111% (range: 107-117%), indicating no substrate effect in the interference-suppressed ADA ELISA.

The drug resistance was evaluated by determining the highest TCZ concentration that a given concentration of positive control ADR could be detected above the cut-point. Table 2b presents a summary of the complete data set.

[Table 2b]

The concentration of anti-IL6R antibody tocilizumab detected in clinical samples is at least 0.5 μg/ml, often in the range of 1 μg/ml to 10 μg/ml. Very low ADA concentrations of 30 ng/ml were detected in the presence of 10 μg/ml TCZ and tested positive. Moreover, the ADA concentrations of 100 ng/ml and 300 ng/ml respectively show drug antibody resistance of 30 μg/ml and even 100 μg/ml. The same experiment carried out by the two-step conventional ADA ELISA used previously for tocilizumab showed more than 10 times higher drug resistance by the interference-suppressed assay (Fig. for 300 ng/ml ADA concentration). See 4).

The concentration of TCZ used as excess free drug in the confirmation assay was based on the data set obtained in the drug interference experiment. To assess the concentration of TCZ capable of inhibiting high levels of ADA in the samples, four different concentrations of positive control samples (1,000 in 100% serum; 500; 250; 83.3 ng/ml) were each added at increasing concentrations. Incubated with TCZ (0 in 100% serum; 16; 31; 63; 125; 250 μg/ml). The TCZ concentration that inhibited more than 95% (corresponding to signal recovery of less than 5%) of the signal measured in the high concentration of the positive control ADA was determined to be 25 μg/ml TCZ.

In order to reduce the likelihood of false-negatives due to differences in the affinity of ADA in the study samples during the test period during the later study, a free drug concentration twice the above measured value was used for further evaluation, i.e. An assay concentration of 40 μg/ml corresponding to 400 μg/ml in 100% serum was used.

The minimum signal inhibition value required to identify specific ADA was determined by pre-incubation of individual blank human serum samples of 16 rheumatoid arthritis patients with TCZ and analyzed in duplicate in one test run. The assay was performed in the presence and absence of 400 μg/ml of free TCZ. The addition of free TCZ was found to reduce the assay signal by an average of 14.1% ranging from -11.5% to 34.8% and an SD of 11.2%. Applying the 99.9% confidence interval (mean +3.09 SD) resulted in a minimal signal reduction of 49% for the blank serum sample. Based on the above calculations, samples were evaluated as positive confirmations when the signal decreased by more than 49% in the presence of excess free drug compared to the case in the absence of free drug. As a reference sample, the corresponding sample without excess TCZ was used. In order to demonstrate the reproducibility of signal inhibition in this confirmatory assay, serum samples with high, medium and low positive QC concentrations were analyzed in triplicate in the presence and absence of a given concentration of excess TCZ.

The interference-suppressed ADA assay for the TCZ had a measurement range of the ADA calibrator from 1,000 ng/ml to 15.6 ng/ml in 100% serum. The intra-analytical precision was less than 5% for all quality controls and the intra-analytical accuracy was 91.1% to 102%. Inter-analytical precision and accuracy were less than 6% and 92.8% to 105%, respectively.

Qualitative analysis of this assay excluded hook and substrate effects.

It can be seen that the drug resistance of the interference-suppressed ADA assay was 10 times higher than that of the previous version.

The confirmation assay was performed essentially as previously described for the screening assay, except that the samples were analyzed in parallel in the absence and presence of excess free drug, ie TCZ. The confirmation capture/detection solution contained an equal volume of capture/detection solution with an additional excess of TCZ (44.4 μg/ml) to achieve a final assay concentration of 40 μg/ml TCZ after addition of the sample. Calculation of percent signal inhibition under confirmed conditions with excess drug was performed using the following formula:

% Signal inhibition = 100 x (1-([AU] drug-pretreated sample/[AU] untreated sample)).

Example 6

Application of interference-suppressed ADA analysis to clinical samples

Means of increasing drug resistance include 1) increasing the concentration of biotinylated and digoxigenylated TCZ (eg up to 1.5 μg/ml); 2) incubating the serum samples simultaneously with TCZ-Bi and TCZ-Dig; 3) prolonged incubation of the serum sample with TCZ-Bi and TCZ-Dig (eg overnight); 4) Instead of a mixture of lysine- and carbohydrate-coupled reagents, only lysine-coupled TCZ-Bi and TCZ-Dig reagents are used; An increased serum substrate content (eg 10% instead of 5%) is used to produce an increase in ADA positivity from 12/28 to 25/28.

Sixteen clinical serum samples were subjected to a series of three different ADA assays: conventional ADA assay, interference-suppressed ADA assay as reported herein without added oligomeric human IgG, and with addition of oligomeric human IgG. Analysis by interference-suppressed ADA analysis as reported in the present invention. The results are summarized in Table 6. Of the 16 samples, 15 tested positive in the version of the ADA assay in which only means 1-5 were performed, whereas interference as reported in the present invention in which means 1-6 were performed as well as conventional assays- In the inhibited drug resistance ADA assay, only 8/16 tested positive and had the same results in 7 of the 8 samples. The seven samples with the same results were characterized by low RF concentrations at the sample and/or baseline. All seven samples contained an ADA of the IgG isotype bound to the Fab portion of tocilizumab, indicating the true tocilizumab specific ADA. Three of the seven samples also had an IgM isotype ADA, but the upper respiratory tract was also bound to the Fab moiety. In contrast, the majority of the remaining samples had an ADA predominantly of the IgM isotype bound to the constant Fc portion of tocilizumab. These samples also contained high RF concentrations, ie >1000 U/ml.

[Table 6]

Using the ADA immunoassay as reported in Example 5, 148 different serum samples from rheumatoid arthritis patients taken after baseline and tocilizumab administration were analyzed. The results of the assay by the interference-suppressed ADA assay were compared with the results obtained by the assay by conventional ADA immunoassay. For a more detailed analysis, a total of 92 serum samples (out of 148 above) from 18 different patients were selected with additional information about the ADA isotype and binding region as well as the clinical event. The patients were selected if they met one or more of the following criteria: 1) ADA positive immune response at any time point; 2) high TCZ serum concentration; 3) Clinical reactions such as infusion-related, hypersensitivity, or hypersensitivity. Analysis of the binding region and isotype of the ADA was performed by a biosensor immunoassay as previously disclosed (Stubenrauch, K., et al., Anal. Biochem. 390 (2009) 189-196). Briefly, the Surface Plasmon Resonance (SPR) assay configuration consists of four parallel flows on a single biosensor chip by immobilization of full-length antibodies and their constant (Fc) and antigen binding (Fab) fragments for differential binding analysis of ADA. Cells were used. Positive control standard conjugates mimicking polyclonal human ADAs of different isotypes were obtained by conjugating polyclonal rabbit antibodies against TCZ to human immunoglobulin (Ig) M, IgG, or IgE (WO 2008/061684). Please refer to). Rheumatoid factor (RF) analysis was performed on a Siemens BN II Nephelometer using RF reagents from Siemens Healthcare Diagnostics (Newark, Delaware). Briefly, polystyrene particles coated with an immuno-complex consisting of human immunoglobulins and anti-human IgG from sheep aggregate upon mixing with samples containing RF. The agglomerates scatter light rays passing through the sample. The intensity of the scattered light is proportional to the concentration of each protein in the sample. The results are evaluated by comparison with a standard of known concentration.

Comparative evaluation of 258 different serum samples from TCZ-treated RA patients, by conventional and interference-suppressed drug tolerance ADA assay as reported herein, showed the same positive results in 12 samples. The conventional analysis measured the positivity of 27 gastric patients, whereas the interference-suppressed drug resistance ADA assay as reported in the present invention measured the positivity of 4 gastric patients. In conclusion, the combination of the means as disclosed herein and the addition of oligomeric human IgG as an ADA assay additive conferred increased drug resistance and interference by suppressed RF compared to conventional ADA assays.

A subset analysis of the data mentioned above is shown in Table 7b below.

[Table 7b]

Study-related CP: 0.215 (conventional ELISA); 0.058 (interference-suppressed ELISA); +: positive ELISA result; -: negative ELISA result

Analysis signals for 27 placebo patients not treated with TCZ in Table 8ab are shown as bars and numbers. Due to the absence of TCZ-treatment induced ADA, a high assay signal in this group is not expected and the signal will indicate potential interference.

[Table 8ab]

Study-related CP: 0.215 (conventional ELISA); 0.058 (interference-suppressed ELISA); +: positive ELISA result; -: negative ELISA result

The signal patterns of both assays are very different: all 27 placebo samples were determined to be positive using conventional ELISA, whereas only 4 of the 27 samples for the interference-suppressed ELISA as reported in the present invention. Only dogs were determined to be positive.

In addition to placebo-treated patients, samples from TCZ-treated patients were analyzed. Table 8b shows the results for the patients prior to TCZ-treatment. Results for TCZ-treated patients are shown in Table 8c.

[Table 8b]

Study-related CP: 0.215 (conventional ELISA); 0.058 (interference-suppressed ELISA); +: positive ELISA result; -: negative ELISA result

[Table 8c]

Study-related CP: 0.215 (conventional ELISA); 0.058 (interference-suppressed ELISA); +: positive ELISA result; -: negative ELISA result

The signal patterns for both assays are not similar: 25 patients were determined to be positive using conventional ELISA, whereas only 10 patients using interference-suppressed ELISA as reported in the present invention. Was determined to be positive.

Example 7

Effect of the types of derivatization of capture and tracer reagents

Single-versus multi-label

The means of increasing drug resistance is to increase the concentration of biotinylated and digoxigenylated TCZ (eg up to 1.5 μg/ml); This could lead to a higher background by sticky digoxigenin; To note the worse drug resistance due to higher cut-points, single biotinylation and single digoxigenation provide a lower background signal by the presence of higher capture and trace concentrations.

Sandwich ELISA was used for both screening and identification of anti-drug antibodies (ADA) against tocilizumab (TCZ) (Stubenrauch, K., et al., Clin. Ther. 32 (2010) 1597-1609). Please refer to). The principle of the method is the capture of ADA in a complex with TCZ-Dig (single) and TCZ-Bi (single), which TCZ-Bi induces immobilization on streptavidin-coated plates. The TCZ-Bi/ADA/TCZ-Dig complex bound to the SA-MTP was detected by the anti-Dig-HRP enzyme conjugated antibody. The principle of the drug-resistant anti-drug antibody assay is shown in FIG. 1. The inclusion of oligomeric IgG as an ADA assay additive results in an interference-suppressed anti-drug antibody assay as shown in FIG. 2. Horseradish peroxidase (HRP) of the polyclonal antibody catalyzes the color reaction of the substrate ABTS. The color intensity is proportional to the concentration of the analyte.

The screening assay was performed at room temperature. Reagents and serum samples were diluted with universal buffer (cat no.4742672), and all washing steps were diluted with washing buffer (PBS, 0.05% Polysorbate 20 (Twin® 20) (cat.no. 11332465-001))). As, it was carried out three times at 300 µl per well. Incubation was carried out under shaking on a microtiter plate shaker (MTP shaker) at 500 rpm. Test samples, QC and CS samples were incubated overnight (16 hours) with capture antibody TCZ-Bi and detection antibody TCZ-Dig. Containing 1.667 μg/ml TCZ-Bi (single) and 1.667 μg/ml TCZ-Dig (single) with oligomeric human IgG ADA assay additive (AAA) in each well of the pre-culture microtiter plate (MTP). 225 μl of the capture/detection solution was loaded and then 25 μl of each sample was added. The production concentrations of TCZ-Bi (single) and TCZ-Dig (single) were 1.5 µg/ml, respectively, and the oligomeric human IgG had a concentration of 50 µg/ml. The loaded MTP was covered to prevent evaporation and incubated overnight. 100 [mu]l of each well of the pre-culture plate was transferred to the wells of a streptavidin-coated microtiter plate (SA-MTP), covered and incubated for 1 hour.

After washing, the polyclonal anti-Dig Fab-HRP conjugate having a concentration of 25 mU/ml was added to each well in a volume of 100 μl and incubated for 1 hour. After washing, the ABTS ready-to-use solution was added to each well in 100 μl aliquots and incubated with shaking for about 10 to 15 minutes. The signal of the color-generating reaction was measured by an ELISA reader at a wavelength of 405 nm (reference wavelength: 490 nm). The absorbance value of each serum sample was measured in duplicate 3 times. The highest standard should reach an optical density (OD) in any unit (AU) of 1.8 to 2.2. The obtained OD data was used to generate a standard calibration curve by non-linear four-parameter matched “Bimmer Rodbart” for calculation of the sample concentration. The sample was found to be positive for ADA if the recovery of this concentration was below the specificity cut-point.

In order to determine the cut-point, intrinsic sera of 35 patients with RD (rheumatic disease) were measured in both assays. As shown in Figures 5 and 6, serum signals in the interference-suppressed anti-drug antibody ELISA compared to the interference-suppressed anti-drug antibody ELISA with TCZ-Bi (single) and TCZ-Dig (single). The fluctuation of the liver is high.

For further evaluation different serum samples from TCZ-treated RA patients were analyzed with both variants of the interference-suppressed ADA-analysis. Since all samples were taken prior to TCZ treatment (baseline), there should be no ADA for TCZ. Signals higher than the cut-point may indicate non-ADA related interference (false positive) to the therapeutic drug (TCZ).

The analysis for multi-labeled TCZ (Figure 7) showed more signal fluctuations than the assay for single labeled TCZ (Figure 8). As can be seen, the variation is not a systematic configuration of the system (which will simply be a movement on the Y-axis), but an increase in the bandwidth of the acquired signal. When using this assay-specific cut-point, almost all samples in the interference-suppressed drug-resistant ADA assay (single) are below the cut-point. Usually all samples measured in the multiplex assay are above the cut-point.

Claims (11)

As a method of detecting an anti-drug antibody against a drug antibody in a sample of a patient with rheumatoid arthritis,
a) a capture reagent and a tracking reagent each having a concentration of at least 0.5 μg/ml are provided;
b) the sample is incubated for 0.5 to 24 hours simultaneously with the capture reagent and the tracer reagent;
c) the capture reagent is obtained by biotinylating only once through a single lysine moiety of the drug antibody to produce a 1:1 conjugate of the drug antibody and biotin, and the tracer reagent comprises a single lysine residue of the drug antibody. Obtained by digoxigenylation via a lysine residue only once to produce a 1:1 conjugate of the drug antibody and digoxigenin;
d) the sample contains at least 1% serum/plasma;
e) oligomeric human IgG is added to the sample prior to incubation with the capture reagent and the tracer reagent;
f) the anti-drug antibody is detected in the complex of the capture reagent and the tracer reagent,
Detection method. The method of claim 1,
The method of detection, wherein the sample comprises an anti-drug antibody and a rheumatoid factor. The method of claim 1,
The detection method, wherein the drug antibody is an anti-inflammatory antibody. The method of claim 3,
The detection method, wherein the anti-inflammatory antibody is an antibody for the treatment of rheumatoid arthritis, juvenile arthritis, or osteoarthritis, or Castleman's disease, or intervascular proliferative glomerulonephritis. The method of claim 1,
The detection method, wherein the drug antibody is an anti-cancer antibody. The method of claim 5,
The detection method, wherein the anticancer antibody is an antibody for the treatment of myeloma or plasmacytoma. The method of claim 1,
The method of detection, wherein the drug antibody is an anti-IL6R antibody. The method of claim 7,
The detection method, wherein the anti-IL6R antibody is tocilizumab. A method of providing data for treating an individual with plasmacytoma, comprising:
The presence of an anti-anti-IL6R antibody (anti-drug antibody) in the sample of the individual to which an anti-IL6R antibody (drug) was administered using the detection method according to any one of claims 1 to 8 was determined. A method comprising detecting. A method of providing data for treating an individual with myeloma, comprising:
The presence of an anti-anti-IL6R antibody (anti-drug antibody) in the sample of the individual to which an anti-IL6R antibody (drug) was administered using the detection method according to any one of claims 1 to 8 was determined. A method comprising detecting. A method of providing data for inhibiting IL6R activity in an individual, comprising:
The presence of an anti-anti-IL6R antibody (anti-drug antibody) in the sample of the individual to which an anti-IL6R antibody (drug) was administered using the detection method according to any one of claims 1 to 8 was determined. A method comprising detecting.

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